Treatment method

ABSTRACT

The invention relates to a method for the treatment of an oestrogen-dependent proliferative disorder of the uterus such as endometriosis and uterine fibroids in a patient, by administering an aromatase inhibitor to the patient intravaginally. This achieves high local levels of aromatase inhibitor Locally, and therefore avoids some of the adverse reactions that are observed when aromatase inhibitors are delivered orally. Further, intravaginal delivery allows inhibition of the local lesional production without significantly affecting the circulating levels which have been produced by the ovaries. This results in minimal side-effects and will allow for longer term treatment than current therapies.

The invention concerns a method for the treatment of anoestrogen-dependent proliferative disorder of the uterus in a patient,by administering an aromatase inhibitor to the patient intravaginally.

All publications, patents and patent applications cited herein areincorporated in full by reference.

The incidence of proliferative disorders of the uterus is very high inwomen of child-bearing age. In particular, conditions known asendometriosis and uterine fibroids affect up to 1 in 4 of women in theWestern world.

Endometriosis is a benign (non-cancerous), chronic condition affectingwomen. It is defined as the presence of both endometrial glandulartissue and stromal cells located outside the uterus. This displacedtissue is known as ectopic endometrium. This disease affects womenduring their childbearing years with deleterious social, sexual andreproductive consequences. Endometriosis has been proposed as one of themost commonly-encountered diseases of gynaecology, with the incidence ofendometriosis in the general population being estimated to be around 5%,although it is thought that it affects 10% to 15% of women between theages of 20 and 45 years. In North America it is estimated that 5.5million women suffer with the condition. In Japan a large surveyconducted by the Ministry of Health in 1997 found that around 130,000women are undergoing hospital treatment for the condition at any onetime, and that the incidence of endometriosis was higher than that forcancer and diabetes. In the United Kingdom, endometriosis causes theloss of 45 days work each year, on average, for every patient and 11% ofsufferers claim state benefit as a direct result of the condition. (DataSources: Pharma Ventures, Eurotech Capital and Bridgehead Technologies)

Endometriosis develops over a number of years until the symptoms aresufficiently severe for the affected patient to seek medical advice. Thecondition is diagnosed by using laparoscopy to visualise theendometriotic deposits. The most common and severe symptom is chronicpelvic pain, as well as dysmenorrhoea (pain in the back and lowerabdomen during periods) and dyspareunia (pain during intercourse). Alink between endometriosis and infertility is evident and between 30%and 40% of women with endometriosis are infertile, although themechanistic link has not been clarified.

The development of endometriosis involves the establishment and growthof endometrial cells at ectopic sites, most commonly the pelvicperitoneum, ovaries and rectovaginal septum, following retrogrademenstruation (see Thomas & Prentice (1992), Repro. Med. Rev., (1):21-36, and Sampson, 1927, Am. J. Obstet. Gynecol., 14: 422469). Thesustained growth of the endometriotic lesions depends on the formationof new blood vessels. Indeed, angiogenic factors, including vascularendothelial growth factor (VEGF), have been detected in the peritonealfluid of women with endometriosis (see, for example, Oosterlynck, etal., 1993, Fertil. Steril., 59: 778-782; European patent applicationEP-A-0 771 192; McLaren, et al., 1996, Hum. Reprod., 11: 200-223; andNisolle. & Donez, 1997, Fertil. Steril., 68,575-596).

Implantation of autologous non-malignant ectopic tissue is a uniquephenomenon suggesting that an abnormal host response may be present inwomen who develop this disease. This theory is supported by the factthat only a minority of women will develop the disease in spite of thecommon occurrence of retrograde menstruation as a source of endometrialtissue.

Endometriosis is now recognised as a multifactorial disease caused bygenetic factors that may determine a woman's susceptibility toendometriosis as well as environmental factors. It has been reportedthat first degree relatives of an affected individual have an increasedrisk of developing endometriosis compared to the general population(see, for example, Kennedy et al., 1995, J. Assist. Reprod. Genetic.,12(1): 32-34; Treolar et al., 1999, Fertil. Steril., 71(4): 701-710; andMalinal et al., 1980, Am. J. Obstet. Gynecol., 137(3): 332-337).

There are many theories proposed for the origin of the disease andvarious cellular and biochemical constituents of the peritoneal fluidhave been reported to play an important role in the pathogenesis of thisdisease. Alterations in multiple aspects of both humoral immunity andcell-mediated immunity have also been reported in suffering individuals(Understanding and Managing Endometriosis. Advances in Research andPractise Ed. A. Lemay & R. Maheux Parthenon Publishing London New York1999).

The growth and development of endometrial tissue appears to depend onthe presence of oestrogen. Drugs that reduce the effect of GnRH (bothagonists and antagonists have this effect) reduce oestrogen levels aswell as levels of progesterones. These drugs, developed initially totreat oestrogen-dependent tumours provide some symptomatic relief inendometriosis. Pregnancy and some oral contraceptive agents also bringabout symptomatic relief. Furthermore, all drugs described to date actby relieving the symptoms of the disease and are not in any sensecurative. This makes a patient permanently dependent on the drug if thesymptoms of disease are to be kept at bay.

Presently, the only treatment of endometriosis that is effective in thelong term involves surgery, although recurrence of the disease oftenoccurs within 5 years post-operation. Certain developments in thetreatment of endometriosis, such as the identification of angiogenicgrowth factors (such as VEGF) which are thought to be partly responsiblefor the establishment and development of endometriosis, have in partpaved the way for the development of new therapeutic agents.Additionally, the discovery of aromatase expression from endometrioticlesions has been suggestive of the use of aromatase inhibitors to treatendometriosis (Bulun et al., 1999, Endocrine Related Cancer, 6:293-301). Indeed, one unusually aggressive case of postmenopausalendometriosis has been successfully treated with an aromatase inhibitor(Bulun et al., 2000, Hum Reprod Update, 6(5):413-8). However, to proceedbeyond this initial work, clinical trials will clearly be required toestablish whether aromatase inhibitors will have a significant role inthe medical management of endometriosis.

Uterine fibroids (also known as leiomyomas, fibromyomas, fibromas,myofibromas and myomas; these terms may be used interchangeably) are asecond form of proliferative disorder of the uterus that afflict a largenumber of women. These benign growths, deriving from the myometrium,consist of masses of smooth muscle fibres and white fibrous tissueswhich can range in size from a small seed to a mass weighing as much asa kilogram. They occur in or on the surface of the uterus, and affectaround 20%-40% of women, generally between the ages of 35 and 45 years.In the United States, Europe and Japan an estimated 3.4 million womenare diagnosed each year with fibroids, of whom a significant numberreceive therapy. According to the Society of Cardiovascular andInterventional Radiology in the USA, 20%-40% of women have fibroids of asignificant size and among African-American women the incidence is ashigh as 50%.

Many women with uterine fibroids are asymptomatic, although manyexperience abnormal uterine bleeding (menorrhagia), pelvic pain, pelvicpressure, urinary frequency or compressive bowel symptoms. Some womenappear to have difficulty becoming pregnant, or/and have an increasedrisk of miscarriage.

The cause of uterine fibroids is not known, but once formed theyestablish a network of new blood vessels that provides the blood supplyto allow them to increase in size.

Uterine fibroids may be treated by conservative surgery, such asendometrial resection of submucous fibroids during hysteroscopy, or bymore invasive surgery such as myomectomy (the surgical removal of thefibroid) and hysterectomy. In the USA, more than 200,000 women undergohysterectomy each year due to fibroids. Less invasive kinds of surgeryare available to remove small fibroids, like hysteroscopy, when a fibreoptic scope is inserted through the vagina to remove them. However,about 10% of women will require repeat surgery as the fibroids re-grow.

Medical treatments of fibroids make use of a variety of agents that mayreduce the symptoms of pain and heavy blood loss as well ascontraceptives and progestogens. A number of women use GnRH agonists toshrink the fibroid but this treatment can only be useful for up to sixmonths and fibroids may re-grow after treatment.

There thus also remains a great need for the discovery of agents witheffective prophylactic or therapeutic value against proliferativedisorders of the uterus, such as endometriosis and uterine fibroids.

SUMMARY OF THE INVENTION

According to the invention, there is provided a method of treating orpreventing an oestrogen-dependent proliferative disorder of the uterusin a patient, by administering an aromatase inhibitor to the patientintravaginally.

The inventors have discovered that the vagina provides an excellentroute for the administration of an aromatase inhibitor to treat orprevent the incidence of oestrogen-dependent proliferative disorders ofthe uterus such as endometriosis and uterine fibroids.

It has been found, through biopsy of endometrial lesions and uterinefibroids, followed by the specific amplification of nucleic acids thatencode aromatase by performing quantitative PCR (QPCR), thatsignificantly elevated levels of aromatase are expressed in endometriallesions and fibroids. Aromatase activity is barely detectable in normal(eutopic) endometrium and myometrium.

This inappropriate aromatase activity gives rise to local biosynthesisof oestrogen. Upregulation of VEGF results from increased oestrogen.This favours the accumulation of oestrogen and VEGF in endometriotictissues, which may contribute to the establishment and growth ofvasculature of the lesions through oestrogen-induced VEGF production.

Additionally, oestrogen-dependent induction of cyclooxygenase gives riseto elevated concentration of prostaglandin E₂ (PGE₂). PGE₂ has beenshown to be a potent stimulator of aromatase expression in endometrioticstromal cells (Noble, L. S. et al. 1997 J. Clin. Endocrinol. Metab. 82No. 2 600-606). Thus, a positive feedback loop is formed and thisfavours continuous production of oestrogen in the endometriotic tissues.Aromatase inhibitors are considered to break this cycle by blockinglocal production of oestrogen in endometriosis. However, theseinhibitors also decrease or block oestrogen formation in other tissues,such as the ovary and subcutaneous fat.

The increased levels of PGE₂ may be responsible for some of the symptomsof endometriosis, such as pain. Inhibition of oestrogen production withan aromatase inhibitor has been noted by Bulun et al to result in areduction in the size of an endometrial lesion in one patient (Takayama,K. et al 1998 Fertil. Steril. 69 No. 4 709-713).

Intrinsic molecular aberrations in pelvic endometriotic implants havebeen previously proposed to contribute significantly to development ofendometriosis. For example, aberrant expression of aromatase, certaincytokines and tissue metalloproteinases, deficiency of17β-hydroxysteroid dehydrogenase (17β-HSD) type 2 and resistance to theprotective action of progesterone are some of these molecularabnormalities (see Khorram et al., 1993, American Journal of Obstetricsand Gynecology, 169: 1545-1549; Sharpe-Timms et al., 1995, Journal ofClinical Endocrinology and Metabolism, 80: 3784-3787; Noble et al.,1996, Journal of Clinical Endocrinology and Metabolism, 81: 174-179;Osteen et al., 1996, Seminars in Reproductive Endocrinology, 15:301-308; Bruner et al., 1997, Journal of Clinical Investigation, 99:2851-2857; Zeitoun et al., 1998, Journal of Clinical Endocrinology andMetabolism, 83: 4474-4480; and Zeitoun et al., 1999, MolecularEndocrinology, 13: 239-253).

A number of studies have demonstrated that oestrogen is produced inendometriotic implants in the pelvis and at other sites through theexpression of the aromatase enzyme, (see Takayama and Bulun, 2001,Nippon Rinsho; 59 Suppl 1:157-60; Kudoh et al., 1997, J Steroid BiochemMol Biol; 63(1-3); 75-80; Bulun et al., 2000, Hum Reprod Update; 6(5):413-8; Kitawaki et al., 2000, J Clin Endocrinol Metab; 85(9): 3292-6;Bulun et al., 2000, J Mol Endocrinol; 25(1):3542; Bulun et al., 1999,Endocr Relat Cancer; 6(2): 293-301; Bulun et al., 2000, TrendsEndocrinol Metab; 11(1): 22-7; Zeitoun et al., 1999, Fertil Steril;72(6): 961-9; Zeitoun et al., 1999, Mol Endocrinol; 13(2): 239-53;Takayama et al., 1998 Fertil Steril; 69(4): 709-13; and Bulun et al.,1997, J Steroid Biochem Mol Biol; 61(3-6): 133-9). Prostaglandins inendometriotic cells have been proposed to stimulate aromatase activityto increase oestrogen biosynthesis. In turn, oestrogen stimulatesprostaglandin formation and the growth of endometriotic implants.

It is also known that endometrial lesions are oestrogen-dependent.During the earlier stages of the development of the lesions, theoestrogen is supplied by the systemic circulation and the source of thehormone is predominantly the ovaries; a small and variable amount ofoestrogen may be produced by adipose tissue. Growth of lesions isthought to relatively slow during the early stages but later a number ofcellular and molecular changes have been documented. Of particularsignificance is the local lesional production of oestrogen. This occurswhen the mesenchymal elements of endometrial lesions begin the aberrantsynthesis of certain enzymes. The local production of oestrogenstimulates growth of glandular tissue and also promotes synthesis ofpain-mediators, especially the prostaglandins. With time, the localproduction of oestrogen becomes more significant thanovarian-synthesized circulating hormone.

It is well established that reduction in ovarian production of oestrogenameliorates the symptoms of endometriosis. However the current methodsfor achieving this involve very significant ovarian suppression tocirculating levels where a number of oestrogen-dependent organs becomecompromised. For example, bone mineral density is critically dependenton adequate levels of oestrogen. For this and other reasons, treatmentwith agents such as the gonadotrophin-releasing hormone agonists canonly be used for six months.

The inventors have now recognized that a significant proportion ofexisting endometrial lesions may be targeted by the transvaginaldelivery of an aromatase inhibitor. This achieves high local levels ofaromatase inhibitor locally, and therefore avoids some of the adversereactions that are observed when aromatase inhibitors are deliveredorally. By transvaginal delivery is meant the movement of aromataseinhibitor molecules from the vaginal cavity into the surrounding tissuesvia a simple diffusion process.

The invention exploits the highly vascularised nature of the vaginalmucosal tissue (which leads to a copious blood supply) to deliver anaromatase inhibitor composition to localised areas and the underlyingtissues that are diseased. Delivery of the aromatase inhibitor throughthe tissue wall is thus fast and this route of administrationfacilitates achieving a concentration of drug that is effective to treator prevent the disorder. A significant effect of the invention is thatintravaginal delivery allows inhibition of the local lesional productionwithout significantly affecting the circulating levels which have beenproduced by the ovaries. This results in minimal side-effects and willallow for longer term treatment than current therapies. The lowering ofthe locally high levels of oestrogen will reduce growth of lesions andalso lower the rates of production of inflammatory mediators which leadto the major symptom of pain.

The vasculature of the female reproductive tract consists of a number ofvascular plexuses of different origin, and there are anastomoses betweena number of the key vessels including the vaginal, ovarian and uterinearteries as well as between the uterine and deep perineal branches ofthe pudendal artery. For example, the arterial supply to the vagina isfrom the vaginal branch of the uterine artery, occasional vaginalbranches of the internal iliac arteries, possible twigs from the middlerectal arteries, and branches from the internal pudendal arteries. Thesevaginal arteries course along the lateral vaginal walls, along the wallsof the uterus, and anastomose with the ovarian artery. The ascendingbranches of the uterine artery lead to the tubal arterial branch andanastomoses with the ovarian artery. Venous return from the vaginalvenous plexus drains into the internal iliac vein, and the uterinevenous return is along uterine veins, which generally parallel thearterial supply. Lymphatic drainage of the vagina drains into theexternal and internal iliac nodes, as well as into the superficialinguinal nodes. Lymphatic drainage of the uterus parallels the uterineblood supply.

The absorption of drugs through the vaginal mucosa and into the systemicvasculature is known, and it has been observed by others that a “uterinefirst pass effect” occurs whereby, for example, the ovarian and uterinetissue levels of some drugs given vaginally are higher than would beexpected from oral administration of the same doses. The exact mechanismof this “first uterine pass” effect is not yet fully understood. Fourhypotheses can be put forth to explain the uterine first pass effect:(1) transvaginally administered drugs may transit to the uterus andother local tissues through the local circulatory system; (2) there maybe direct diffusion of drug into the uterus and other local tissues; (3)drugs may reach the uterus through the lymphatics; or (4) a“counter-current” redistribution of drug may occur between arteries andveins.

By an “oestrogen-dependent proliferative disorder of the uterus” ismeant any oestrogen-dependent non-malignant disorder that occurs infemales that stems from uterine tissue. Particular examples ofoestrogen-dependent proliferative disorders of the uterus that areincluded within the terms of the invention are endometriosis and uterinefibroids. Humans are preferred patients for treatment, althoughnon-human mammals, such as domesticated and companion animals, may alsobe treated.

Endometriosis is the name given for the occurrence of endometriumtissue, found at ectopic sites in the body. Endometriotic lesions may bedetermined histologically using markers or by looking for endometrialglands and stromal elements in tissue at ectopic sites. Although thistissue type may be in any anatomical location, it is generally to befound in the region of the ovary, peritoneum, or recto-vagina and it isendometriotic lesions in these locations that may be particularlybeneficially treated according to the method of the present invention.

Uterine fibroids, or uterine leiomyomas are a second oestrogen-dependentproliferative disorder of the uterus, marked by the presence of one ormore benign tumours found consisting of connective tissue and musclefound on the uterine wall. Fibroids may lie just below the uterinelining or near the uterus' outer covering, whilst others are locatednear the cervix, or close to the openings of the fallopian tubes. Theyare usually detected on abdominal and pelvic examination as well as oninternal examination, which may reveal the uterus to be enlarged and/ordetect the fibroid as a smooth, firm lump. Ultrasound is the most usefultest for diagnosing fibroids as these tumours have a characteristicappearance that distinguishes them from pelvic cysts.

According to the invention, an aromatase inhibitor should be deliveredintravaginally. By “intravaginally” is meant that an aromatase inhibitoris administered via the vagina, such that the inhibitor crosses thevaginal mucosa to enter the blood and/or lymphatic system by way oflocal absorption though the highly vascularised tissue in this area.

Vaginal administration of an aromatase inhibitor for the treatment ofendometriosis and fibroids is anticipated to lead to both improvedefficacy, and to a reduced adverse effects profile compared to oraltherapy.

Of great significance is the fact that an effective concentration ofaromatase inhibitor may effectively be delivered to the diseased tissueitself, i.e. the endometrial lesion or uterine fibroid, both of whichdisorders generally present in tissues or organs that are close to thevagina, such as in the vaginal tissues, uterus, ovaries and fallopiantubes, rectovaginal region and cul-de-sac and other tissues and organswithin the peritoneal cavity. In addition, such levels of aromataseinhibitor are expected to affect newly-shed endometrial cells travellingup the fallopian tubes and into the peritoneum, reducing their rate ofgrowth and the probability of implantation.

The oral delivery of a drug such as an aromatase inhibitor may involvethe destruction of the aromatase inhibitor by local conditions such asthose that are encountered in the stomach. First-pass metabolism by theliver is also a problem suffered by the oral delivery route.Furthermore, in order to achieve a concentration of aromatase inhibitorthat is effective at the diseased tissue, a large dose must beadministered that then leads to high systemic levels of inhibitorcompound. The oral use of aromatases to treat metastatic breast cancerhas been associated with a variety of adverse events including hotflushes, dizziness, oedema, sweating, nausea, vomiting, chest or backpain, fatigue, headache, insomnia, dyspnoea, asthenia, emotionallability, and depression.

By administering the aromatase inhibitor via the vaginal route, a muchlower concentration of inhibitor compound needs to be used than would benecessary if the inhibitor were to be administered parenterally, forexample, by the oral route. The method of the invention yieldssignificant local levels of aromatase inhibitor while maintainingcirculating blood levels that are low enough to avoid most undesiredside effects. This means that susceptible tissues will be exposed to amuch lower concentration of inhibitor, whilst diseased areas will beexposed to a much higher concentration of inhibitor than would beachieved using any other method of administration.

The oral route is also not suitable for certain types of drugcompound—in order to be effectively absorbed into the blood, anorally-delivered drug compound must be orally-active, which means thatit is either passively transported or actively transported. Only a smallproportion of drug molecules fulfil all these criteria, which thuslimits the number of drug compounds that might be used to treat anoestrogen-dependent proliferative disorder of the uterus. The oral routeis also precluded when vomiting has occurred, or is likely to occur, orwhen the patient is unable to swallow successfully.

Intra-vaginal devices are known that are suitable for the delivery ofaromatase inhibitors intravaginally, according to the method of theinvention. Suitable devices include those of the type where a medicamentis impregnated into the device, and of the type that carries anencapsulated medicament.

Movement of pharmaceutical molecules from the vaginal cavity into thesurrounding tissues will generally occur via a simple diffusion process.Net diffusion may be given by the equation:Net diffusion=k.D.(C _(vag) −C _(tiss));where k is a constant, D is the diffusion constant for the molecule,C_(vag) is the molecular concentration in the vagina at the surface ofthe mucosa and C_(tiss) is the molecular concentration in the tissuesurrounding the vagina. In order to achieve maximum rates of uptake ofpharmaceuticals from the vaginal cavity across the vaginal mucosa andinto the surrounding tissues and body fluids, C_(vag) should bemaintained at as high a level as possible.

Examples of devices suitable for the intravaginal delivery of anaromatase inhibitor include those described in U.S. Pat. No. 4,309,997,U.S. Pat. No. 4,318,405, U.S. Pat. No. 5,273,521, U.S. Pat. No.5,299,581, GB 1,581,474, U.S. Pat. No. 5,954,688, U.S. Pat. No.4,402,693, U.S. Pat. No. 3,948,265, U.S. Pat. No. 6,086,909, U.S. Pat.No. 3,545,439, U.S. Pat. No. 3,902,493, U.S. Pat. No. 2,739,593, U.S.Pat. No. 3,521,637, U.S. Pat. No. 3,884,233, U.S. Pat. No. 4,286,596,U.S. Pat. No. 6,197,327, U.S. Pat. No. 5,527,534, EP-A-0,703,802, GB2,069,336, U.S. Pat. No. 4,601,714, U.S. Pat. No. 5,299,581, U.S. Pat.No. 6,159,174, WO99/18884, WO99/40966, W000/48539 and co-owned,co-pending International patent application PCT/GB01/01789. Otherexamples of suitable devices will be clear to those of skill in the art.

The aromatase inhibitor may also be administered intravaginally as abioadhesive formulation, for example, in the form of a gel, cream,tablet, pill, capsule, suppository, film, or any other pharmaceuticallyacceptable form that remains in the vaginal cavity and does not washaway easily. If applied as a bioadhesive formulation, the formulationshould remain attached to the epithelial surfaces of the vaginal mucosafor a significant period, at the minimum, for example, between about 30minutes to twenty-four hours. This preferred level of bioadhesion mayadvantageously be attained by the inclusion of a bioadhesive agent inthe pharmaceutical formulation, such as a cross-linking agent, so thatan appropriate level of bioadhesion results. Suitable formulations aredescribed, for example, in U.S. Pat. No. 4,615,697.

Suitable aromatase inhibitors for use in the methods of the presentinvention include any compound that inhibits the formation of oestrogensfrom their precursors by an aromatase enzyme. Such compounds can be usedeither alone or in combination with other aromatase inhibitor compounds.Certain classes of suitable aromatase inhibitors include non-steroidal,weak steroid and steroidal aromatase inhibitors.

Examples of aromatase inhibitors that are suitable for use in thepresent invention include anastrozole (Armimidex); letrozole;exemestane; vorozole; YM 511 (Yamanouchi Pharmaceutical); YM 553(Yamanouchi Pharmaceutical); [(4-bromobenzyl)(4-cyanophenyl)amino]azolesand their azine analogs; 4-N-substituted amino-4H-1,2,4-triazolederivatives; 3-[N-(2-chlorobenzyl)amino]-6-(1H-imidazol-1-yl)pyridazinedihydrochloride (CAS 124070-28-3, MFI-279); aminoglutethimide;4-hydroxy-androstenedione; 4-hydroxy-4 -androstene-3,17-dione;4-acetoxy-4-androstene-3,17-dione; fadrozole hydrochloride (CGS 16949A)(Bonzol; Mitsubishi-Tokyo Pharmaceuticals Inc); formestane;1-methylandrosta-1,4-diene-3,17-dione;1-methylandrosta-1,4-diene-3,17-dione (described in German patentapplication 3,322,285); testolactone(17a-oxa-D-homoandrosta-0,1,4-diene-3,17-dione) (described in “Journalof Clinical Endocrinology and Metabolism”, (1979) 49: 672);androsta-4,6-diene-3,17-dione; androsta-4,6-dien-17.beta.-ol-3-oneacetate; androsta-1,4,6-triene-3,17-dione;4-androstene-19-chloro-3,17-dione; 4-androstene-3,6,17-trione; compoundsdescribed in “Endocrinology” (1973), vol. 92(3): 874; the 19-alkynylatedsteroids disclosed in German patent application 3,124,780; the10-(1,2-propadienyl) steroids described in German patent application3,124,719; the 19-thioandrostane derivatives described inEP-A-0,100,566; 4-androsten4-ol-3,17-dione (described in “Endocrinology”1977, 100(6): 1684 and in U.S. Pat. No. 4,235,893), and its esters; the1-methyl-15.alpha.-alkyl-androsta-1,4-diene-3,17-diones described inGerman patent application 3,539,244; the10.beta.-alkynyl-4,9(11)-estradiene derivatives described in Germanpatent application 3,644,358;1,2.beta.-methylene-6-methylene-4-androstene-3,17-dione (described inEP-A-0 250 262); or mixtures of any of these aromatase inhibitorcompounds.

A number of references detail the effect of aromatase inhibitors such asthose listed above in inhibiting oestrogen production, including thefollowing: Kudoh et al., 1997, J Steroid Biochem Mol Biol, 63(1-3):75-80; Okada et al., 1997, Chem Pharm Bull (Tokyo), 45(8): 1293-9; Okadaet al., 1997, Chem Pharm Bull (Tokyo), 45(3): 482-6; Okada et al., 1996,Chem Pharm Bull (Tokyo), 44(10):1871-9; Kudoh et al., 1996, J SteroidBiochem Mol Biol, 58(2): 189-94; Kudoh et al., 1995, J Steroid BiochemMol Biol, 54(5-6): 265-71.

The dosage of the aromatase inhibitor that is administered should betherapeutically-effective, i.e. a dosage amount that is necessary totreat, ameliorate, or prevent the oestrogen-dependent proliferativedisorder of the uterus, or to exhibit a detectable therapeutic orpreventative effect. This dosage will vary depending on various factorssuch as the potency of the inhibitor compound, its toxicity in thetreated patient, the general health, age and weight of the patient, thehormonal levels of the patient, the patient's diet, the time andfrequency of administration, drug combination(s), reactionsensitivities, tolerance/response to therapy, the stage of the disease,the degree of spread of diseased tissue, the lifetime of the inhibitorcompound in its active state, the solubility of the compound, itsabsorption characteristics across the vaginal mucosa, the eventualtissue concentration to be attained and so on. [This dosage amount canbe determined by routine experimentation and is within the judgement ofthe clinician.]

For any aromatase inhibitor compound, a therapeutically effective dosagecan be estimated initially either in cell culture assays, for example,endometrial or smooth muscle cells from fibroids of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs, primates such asbaboons, macaques, and so on. The animal model may also be used todetermine the appropriate concentration range for administration. Suchinformation can then be used to determine useful dosages for humans.

Generally, the amount administered in one dose will be between 100 μgand 1 g, preferably between 100 μg and 10 mg of an aromatase inhibitorsuch as 1-methylandrosta-1,4-diene-3,17-dione, or abiologically-equivalent dose of any other aromatase inhibitor as listedabove. The amount selected will depend, of course, on the dosageprescribed, but undesirably high dosages may advantageously be avoidedby using intravaginal delivery, as the concentration of inhibitorcompound in the vicinity of the vaginal tissue wall is maintained at ahigh level.

The frequency of dosage may also be varied so as to administer anaromatase inhibitor most effectively. Conveniently, the dose may berepeated either daily, weekly, monthly, or quarterly (three monthly). Ofcourse, the actual amount that is administered will be altered to takethe dosage frequency into account.

In order to be administered intravaginally, the aromatase inhibitorshould preferably be administered as part of a pharmaceuticalformulation, including a pharmaceutically-acceptable carrier. Suchcarriers include large, slowly metabolised macromolecules such asproteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, polyethylene glycol, PDMS,microspheres, hydrogels, and inactive virus particles, provided that thecarrier does not itself induce the production of antibodies harmful tothe individual receiving the composition, and which may be administeredwithout undue toxicity. Pharmaceutically acceptable salts can also beused, for example, mineral acid salts such as hydrochlorides,hydrobromides, phosphates, sulphates, and the like; and the salts oforganic acids such as acetates, propionates, malonates, benzoates, andthe like. A thorough discussion of pharmaceutically acceptable carriersis available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J.1991).

Pharmaceutically acceptable carriers in the pharmaceutical formulationsof the invention may additionally contain liquids such as water, saline,glycerol and ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, pH buffering substances, lubricants,plasticizing agents, preservatives, gel formers, tablet formers, pillformers, suppository formers, film formers, cream formers,disintegrating agents, coatings, binders, vehicles, colouring agents,taste and/or odour controlling agents, humectants, viscosity controllingagents, pH-adjusting agents, absorption enhancers, and the like, may bepresent in such compositions.

According to a further aspect of the invention, there is provided apharmaceutical formulation comprising an aromatase inhibitor compound,for use in the treatment of an oestrogen-dependent proliferativedisorder of the uterus by intravaginal administration.

The invention also provides the use of an aromatase inhibitor compoundin the manufacture of a medicament for the treatment of anoestrogen-dependent proliferative disorder of the uterus by intravaginaladministration.

The invention also provides an intra-vaginal device comprising anaromatase inhibitor compound according to any one of the embodiments ofthe invention described above, in a therapeutically-effective amount.The inhibitor compound may be coated onto the intra-vaginal device,impregnated or absorbed into the device, or applied to the device by anysuitable means that allows the compound to be attached or bonded to thedevice, yet which allows the compound to be available for absorptioninto the vaginal mucosa, as will be clear to those of skill in the art.A particular preferred example of a suitable intra-vaginal device isthat described in co-pending, co-owned International patent applicationPCT/GB01/01789.

Various aspects and embodiments of the present invention will now bedescribed in more detail by way of example. It will be appreciated thatmodification of detail may be made without departing from the scope ofthe invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-C: Comparison of aromatase transcript levels in human eutopicand ectopic endometrium in 11 patients suffering from endometriosis.Aromatase levels of eutopic endometrium are shown by open bars and thelevels in endometriotic lesions by filled bars. Table 1 provides anexplanation of the nomenclature used in these Figures.

FIG. 2: Comparison of aromatase transcript in human myometrium anduterine fibroid tissue (QPCR TaqMan data).

FIG. 3 shows levels of radioactive counts in endometrial lesions in ababoon model following intra-vaginal administration of a radiolabelledaromatase inhibitor.

FIG. 4: A: Laparoscopic aspect of lesion 5 in the medial anteriorbladder of baboon PAN2615. B: Laparoscopic aspect of lesion 10 in theposterior uterus of baboon PAN2615.

FIG. 5: A. and B: Histological confirmation of the presence ofendometrial glands in lesion 13 of baboon PAN2615. C: Histologicalconfirmation of the presence of glands in eutopic endometrium of baboonPAN2615. The presence of spiral arterioles is also shown.

EXAMPLE 1 Detection of Aromatase Transcript in Human Myometrium andUterine Fibroid Tissue

RNA isolation from fresh tissues was performed using the TRIZOL ReagentMethod for the myometrial and fibroid samples and the QIAGEN Midi RNeasykit for all endometrial samples. For the myometrial and fibroid samples,1 μg of RNA was reverse-transcribed to cDNA using the Superscipt FirstStrand Synthesis Kit (GIBCO BRL, Life Technologies) and random primers.0.1 and 2 μg of RNA were used for reverse transcription of theendometrial samples. The cDNA quality was validated by amplification ofthe GAPDH gene and the absence of genomic DNA was confirmed by usingcalbindin specific primers in exon 1 and 2 of the calbindin gene.(CALB-FOR: GACACACACCCCGCTGTAC; CALB-REV: TGCTGGAGCTCCTGGATC)

For the endometrial and endometriosis samples, undiluted cDNA was usedfor the Q-PCR experiments, while 1:10 dilution of cDNA was used for allthe myometrial and fibroid samples.

Primers and a probe specific for the aromatase gene were designed usingthe Primer Express Software. The probe was labelled with FAM dye(Xmax=518 nm). The sequences of primers and the probe are shown below:Aromatase Forward CCCCGGCCTTGTTCGT Aromatase ReverseCCTCCAACCTGTCCAGATGTGT Aromatase Probe TGGTCACAGTCTGTGCTGAATCCCTCAA

The primer concentrations were optimised in order to determine theminimum primer concentration for obtaining maximum signal. The PCRreaction mix used for primer optimisation consisted of 25 μl of TaqManUniversal PCR Master Mix (2×), 5 μl of Forward primer (50-900 nM), 5 μlof Reverse primer (50-900 nM), 5 μl of 2 μM TaqMan probe, 5 μl of DNAsample and 5 μl of water. The thermal cycling conditions for primer andprobe optimisation are described below: 50° C.  2 min  1 cycle 95° C. 10min  1 cycle 95° C. 15 sec 40 cycles 60° C.  1 min

The probe concentration was optimised by using the conditions describedfor the primer optimisation, where the forward and reverse primers areused at their optimal concentrations.

All assays were then performed using optimum primer and probeconcentrations and thermal cycling conditions shown below: PCR reactionmix AmpliTaq Master Mix 12.5 μl Forward primer  300 nM Reverse primer 300 nM Probe (1 μM stock)  2.5 μl Thermocycling conditions 50° C.  2min  1 cycle 95° C. 10 min  1 cycle 95° C. 15 sec 35 cycles 60° C.  1min

All experiments were performed in triplicate and all samples werenormalised by using as endogenous control, 18S mRNA, to account fordifferences in the amount of total RNA added to each reaction. The 18SQ-PCR was performed using the exact same dilution and quantity of cDNAas used for the aromatase Q-PCR and following the protocol described inthe PE Biosystem's pre-developed assay. Human placenta RNA was used asthe reference sample for allowing comparisons between different samples.

Two transcript variants of aromatase exist, variant 1 (GeneBank entryNM-000103) and variant 2 (GeneBank entry NM-031226) which has a longer5′ UTR than variant 1. Both variants encode the same protein. The PCRprimers used in this invention were specific for regions common to bothvariants.

Raised levels of aromatase transcript were observed in the endometrioticlesions of all 11 endometriosis patients tested. The relative amount ofaromatase transcript was patient-dependent with endometriotic lesionsfrom some patients having as much as several thousand-fold higher levelswhen compared to the eutopic endometrium (H) from the same patient (suchas in patients S and Q). Aromatase transcript was barely detectable inthe eutopic endometrium of all patients tested.

Most interestingly, aromatase transcript was upregulated in lesionslocated at different regions of the patient's peritoneal cavity whetherthey were found on uterosacral ligament, (SU) retovaginal septum (RFS),ovaries (V) or/and pelvic side wall (PSWL) (FIG. 1).

High levels of aromatase transcript were also observed in all fibroidspecimens tested. Aromatase was barely detectable in matched myometriumfrom the same patients. 40% of the fibroids tested had levels higherthan 50-fold when compared to their corresponding myometrium (FIG. 2).TABLE 1 Nomenclature used in FIGS. 1 A-C. UH Patient U, eutopic(healthy) endometrium URVS Patient U, endometriotic lesion isolated fromrectovaginal septum SH Patient S, eutopic (healthy) endometrium SRVSPatient S, endometriotic lesion isolated from rectovaginal septum QHPatient Q, eutopic (healthy) endometrium QVV Patient Q, endometrioticlesion isolated from vaginal vault QPSWL Patient Q, endometriotic lesionisolated from pelvic side wall KH Patient K, eutopic (healthy)endometrium KUS Patient K, endometriotic lesion isolated fromuterosacral ligament RH Patient R, eutopic (healthy) endometrium RRVSPatient R, endometriotic lesion isolated from rectovaginal septum RPFPatient R, endometriotic lesion isolated from posterior formix IHPatient I, eutopic (healthy) endometrium IV Patient I, endometrioticlesion isolated from ovary TH Patient T, eutopic (healthy) endometriumTPSWL Patient T, endometriotic lesion isolated from pelvic side wall PHPatient P, eutopic (healthy) endometrium PVL Patient P, endometrioticlesion isolated from left ovary PVR Patient P, endometriotic lesionisolated from right ovary LH Patient L, eutopic (healthy) endometriumLRV Patient L, endometriotic lesion-1-isolated from rectovaginal regionLR Patient L, endometriotic lesion isolated from the rectal region NHPatient N, eutopic (healthy) endometrium NVR Patient N, endometrioticlesion isolated from right ovary OH Patient O, eutopic (healthy)endometrium OV Patient O, endometriotic lesion isolated from ovary

Example 2 Treatment of Endometrial Lesions in Baboons

A condition that closely resembles endometriosis in humans can begenerated in baboons. In this model endometrial tissue is removed bybiopsy during menstruation. This tissue is then introduced into theperitoneum of the same animal using a laporoscopic procedure.Endometrial lesions can begin to develop. If the procedure is repeatedin the same animals at the time of subsequent menses then most if notall will develop advanced lesions. The basic techniques are described inmore detail in D'Hooghe T. M. (1997) Fertil. Steril. 68 (4): 613-625 andFazleabas A. T. (2002) Ann. NY Acad. Sci. 955: 308-317.

Endometriosis is thought to develop when fragments of endometrial tissuethat are shed during the menstrual process, rather than passing outthrough the vagina, move retrogradely along the Fallopian tube andeventually enter the peritoneal cavity. The animal model used heremimics this process by directly introducing the endometrial tissue intothe peritoneum.

Endometriosis was induced in 3 baboons as follows. All three baboonswere initially evaluated by laparoscopy for absence of lesions andadhesions. After the animals were confirmed to be clear of disease,endometrium was obtained during the first 2 days of menstruation bycurettage from each animal and seeded back into the peritoneal cavity ofthe animal (first inoculation). At the next menses, a second evaluationlaparoscopy was performed to document the presence of lesions andadhesions and the animals were inoculated for a second time withmenstrual endometrium (second inoculation). One month after the secondinoculation, all three animals were evaluated by laparoscopy for thepresence of lesions and adhesions and disease was confirmed in all threeanimals. FIG. 4 shows in panel A a laparoscopic aspect of a particularlesion (lesion 5) in the medial anterior bladder of baboon PAN2615,whilst panel B shows a laparoscopic aspect of lesion 10 in the posterioruterus of the same baboon. FIG. 5 shows histological confirmation of thepresence of endometrial glands in lesion 13 of baboon PAN2615 (panels Aand B) and confirmation of the presence of glands in eutopic endometriumof this baboon (panel C). The presence of spiral arterioles is shown.

The timing of all procedures is shown below: Aromatase First inoculationSecond inoculation administration Animal 1 Day 1 Day 36 Day 99 Animal 2Day 1 Day 36 Day 107 Animal 3 Day 1 Day 35 Day 108

The aromatase inhibitor used in this study was C14-labelledaminoglutethimide.

“D,L”-3-“4-Aminophenyl”-3-ethyl-2,6-piperidinedione[Phenyl-14C“U”](aminoglutethamide) was dissolved in the minimal volume of dimethylsulphoxide (DMSO). Specific activity was 57.8 mCi/mmol and radiochemicalpurity was >99% on HPLP. This solution was diluted into glycerol toprovide the required concentrations and these resulting mixturesadministered intra-vaginally to the anaesthetized animals. Systemicblood samples were taken at time intervals and at three hours, biopsiesof endometrial lesions (and other tissue) were extirpated forradioactive counting.

Histology reveals significant variation between individual lesions and,not surprisingly, there are differences in the levels of radioactivityfound in individual lesions. Some lesions will not be detected due totheir anatomical location. In this study doses of 0.3, 0.6 and 0.9 mCiwere administered to the individual animals and the numbers of obviouslesions identified were 1, 3 and 4 respectively.

The results shown in FIG. 3 clearly show there has been transfer ofaromatase inhibitor from vagina to endometrial lesions. Although bloodlevels were detectable and there was a dose-response relationship,levels never exceeded 2 c.p.m. per microgram confirming that thelabelling of endometrial lesions has occurred via a direct drainage fromvaginal mucosa rather than via systemic circulation. The fact thataromatase inhibitor was targetted to these lesions means that treatmentat these sites was successfully effected.

1. A method of treating or preventing an oestrogen-dependentproliferative disorder of the uterus in a patient comprisingadministering to the patient an aromatase inhibitor intravaginally.
 2. Amethod according to claim 1, wherein the oestrogen-dependentproliferative disorder of the uterus is endometriosis.
 3. A methodaccording to claim 1, wherein the oestrogen-dependent proliferativedisorder of the uterus is uterine fibroids.
 4. A method according toclaim 1, wherein said aromatase inhibitor compounds is deliveredintravaginally using an intra-vaginal delivery device.
 5. A methodaccording to claim 1, wherein said aromatase inhibitor compound isadministered as part of a pharmaceutical formulation.
 6. A methodaccording to claim 5, wherein the pharmaceutical formulation contains abioadhesive agent and/or absorption enhancer.
 7. A method according toclaim 1, wherein said aromatase inhibitor is a compound that inhibitsthe formation of an oestrogen from its precursor by an aromatase enzyme.8. A method according to claim 7, wherein said aromatase inhibitor isselected from the group consisting of anastrozole; letrozole;exemestrane; vorozole; YM 511 (Yamanouchi Pharmaceutical); YM 553(Yamanouchi Pharmaceutical); [(4-bromobenzyl)(4-cyanophenyl)amino]azolesand their azine analogs; 4-N-substituted amino-4H-1,2,4-triazolederivatives; 3-[N-(2-cholorbenzyl)amino]-6-(1H-imidazol-1-yl)pyridazinedihydrochloride (CAS 124070-28-3, MFF-279); aminoglutethimide;4-hydroxy-androstenedione; 4-hydroxy-4-androstene-3,17-dione;4-acetoxy-4-androstene-3,17-dione; fadrozole hydrochloride (CGS 16949A)(Bonzol; Mitsubishi-Tokyo Pharmaceuticals Inc); formestane;1-methylandrosta-1,4-diene-3,17-dione; 1-methylandrosta-1,4-diene-3,17-dione 17a-oxa-D-homooandrosta-1,4-diene-3,17-dione;androsta-4,6-diene-3,17-dione; androsta-4,6-dien-17.beta.-ol-3-oneacetate; androsta-1,4,6-triene-3,17-dione;4-androstene-19-chloro-3,17-dione; 4-androstene-3,6,17-trione; compoundsdescribed in “Endocrinology” (1973), vol. 92(3): 874; the 19-alkynylatedsteroids disclosed in German patent application 3,124,780; the10-(1,2-propadienyl) steroids described in German patent application3,124,719; the 19-thioandrostane derivatives described inEP-A-0,100,566; 4-androsten-4-ol-3,17-dione and its esters; the1-methyl-15.alpha.-alkyl-androsta-1,4-diene-3,17-diones described inGerman patent application 3,539,244; the10.beta.-alkynyl-4,9(11)-estradiene derivatives described in Germanpatent application 3,644,358;1,2.beta.-methylene-6-methylene-4-androstene-3,17-dione; or mixtures ofany of these aromatase inhibitor compounds.
 9. A method according toclaim 1, wherein the amount of aromatase inhibitor that is administeredin one dose is between 100 μg and 1_(g), preferably between 100 μg and10 mg.
 10. A method according to claim 9, wherein the frequency ofdosage is either daily, weekly, monthly, or quarterly (three monthly).11. A pharmaceutical formulation comprising an aromatase inhibitorcompound as recited in claim 1, for use in the treatment of anoestrogen-dependent proliferative disorder of the uterus by intravaginaladministration.
 12. (canceled)
 13. An intra-vaginal device comprising anaromatase inhibitor compound in a therapeutically-effective amount.